Bottom Line:
Mycobacterial FASII undertakes mycolic acid biosynthesis, which relies on a set of essential enzymes, including 3-oxoacyl-AcpM reductase FabG1/Rv1483.Thereafter, FabG2-FabG5 were expressed as mitochondrial proteins in the oar1Delta strain, and FabG4 was found to complement the mutant phenotype and contain high levels of 3-oxoacyl-thioester reductase activity.Hence, like FabG1, FabG4 is also an essential, physiologically functional 3-oxoacyl-thioester reductase, albeit the latter's involvement in mycobacterial FASII remains to be explored.

ABSTRACTMycobacterium tuberculosis represents a severe threat to human health worldwide. Therefore, it is important to expand our knowledge of vital mycobacterial processes, such as that effected by fatty acid synthase type 2 (FASII), as well as to uncover novel ones. Mycobacterial FASII undertakes mycolic acid biosynthesis, which relies on a set of essential enzymes, including 3-oxoacyl-AcpM reductase FabG1/Rv1483. However, the M. tuberculosis genome encodes four additional FabG homologs, designated FabG2-FabG5, whose functions have hitherto not been characterized in detail. Of the four candidates, FabG4/Rv0242c was recently shown to be essential for the survival of M. bovis BCG. The present work was initiated by assessing the suitability of yeast oar1Delta mutant cells lacking mitochondrial 3-oxoacyl-ACP reductase activity to act as a surrogate system for expressing FabG1/MabA directed to the mitochondria. Mutant yeast cells producing this targeted FabG1 variant were essentially wild type for all of the chronicled phenotype characteristics, including respiratory growth on glycerol medium, cytochrome assembly and lipoid acid production. This indicated that within the framework of de novo fatty acid biosynthesis in yeast mitochondria, FabG1 was able to act on shorter (C(4)) acyl substrates than was previously proposed (C(8-20)) during mycolic acid biosynthesis in M. tuberculosis. Thereafter, FabG2-FabG5 were expressed as mitochondrial proteins in the oar1Delta strain, and FabG4 was found to complement the mutant phenotype and contain high levels of 3-oxoacyl-thioester reductase activity. Hence, like FabG1, FabG4 is also an essential, physiologically functional 3-oxoacyl-thioester reductase, albeit the latter's involvement in mycobacterial FASII remains to be explored.

Mentions:
To demonstrate more accurately that expression of mitochondrial versions of FabG1 or FabG4 in the oar1Δ mutant cells could compensate for the missing activity attributed to native Oar1p, the four strains (each expressing FabG1, FabG4, Oar1p or Cta1p) were propagated overnight in liquid SD-Ura medium. Following tenfold serial dilution, cultures were spotted onto solid SD-Ura (glucose) or SCglycerol (glycerol) media, and the plates were incubated at 30°C until single colonies were detectable (Fig. 2). As anticipated, mutant oar1Δ cells expressing Oar1p could form single colonies on glycerol, whereas those cells expressing Cta1p were not able to proliferate on this medium, vindicating the experimental design. Importantly, the results demonstrated that the respiratory deficient phenotype of the oar1Δ mutant could be rescued by FabG1 and FabG4, indicating functional complementation.Fig. 2

Mentions:
To demonstrate more accurately that expression of mitochondrial versions of FabG1 or FabG4 in the oar1Δ mutant cells could compensate for the missing activity attributed to native Oar1p, the four strains (each expressing FabG1, FabG4, Oar1p or Cta1p) were propagated overnight in liquid SD-Ura medium. Following tenfold serial dilution, cultures were spotted onto solid SD-Ura (glucose) or SCglycerol (glycerol) media, and the plates were incubated at 30°C until single colonies were detectable (Fig. 2). As anticipated, mutant oar1Δ cells expressing Oar1p could form single colonies on glycerol, whereas those cells expressing Cta1p were not able to proliferate on this medium, vindicating the experimental design. Importantly, the results demonstrated that the respiratory deficient phenotype of the oar1Δ mutant could be rescued by FabG1 and FabG4, indicating functional complementation.Fig. 2

Bottom Line:
Mycobacterial FASII undertakes mycolic acid biosynthesis, which relies on a set of essential enzymes, including 3-oxoacyl-AcpM reductase FabG1/Rv1483.Thereafter, FabG2-FabG5 were expressed as mitochondrial proteins in the oar1Delta strain, and FabG4 was found to complement the mutant phenotype and contain high levels of 3-oxoacyl-thioester reductase activity.Hence, like FabG1, FabG4 is also an essential, physiologically functional 3-oxoacyl-thioester reductase, albeit the latter's involvement in mycobacterial FASII remains to be explored.

ABSTRACTMycobacterium tuberculosis represents a severe threat to human health worldwide. Therefore, it is important to expand our knowledge of vital mycobacterial processes, such as that effected by fatty acid synthase type 2 (FASII), as well as to uncover novel ones. Mycobacterial FASII undertakes mycolic acid biosynthesis, which relies on a set of essential enzymes, including 3-oxoacyl-AcpM reductase FabG1/Rv1483. However, the M. tuberculosis genome encodes four additional FabG homologs, designated FabG2-FabG5, whose functions have hitherto not been characterized in detail. Of the four candidates, FabG4/Rv0242c was recently shown to be essential for the survival of M. bovis BCG. The present work was initiated by assessing the suitability of yeast oar1Delta mutant cells lacking mitochondrial 3-oxoacyl-ACP reductase activity to act as a surrogate system for expressing FabG1/MabA directed to the mitochondria. Mutant yeast cells producing this targeted FabG1 variant were essentially wild type for all of the chronicled phenotype characteristics, including respiratory growth on glycerol medium, cytochrome assembly and lipoid acid production. This indicated that within the framework of de novo fatty acid biosynthesis in yeast mitochondria, FabG1 was able to act on shorter (C(4)) acyl substrates than was previously proposed (C(8-20)) during mycolic acid biosynthesis in M. tuberculosis. Thereafter, FabG2-FabG5 were expressed as mitochondrial proteins in the oar1Delta strain, and FabG4 was found to complement the mutant phenotype and contain high levels of 3-oxoacyl-thioester reductase activity. Hence, like FabG1, FabG4 is also an essential, physiologically functional 3-oxoacyl-thioester reductase, albeit the latter's involvement in mycobacterial FASII remains to be explored.